oligo dt primer  (Qiagen)

 
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    Oligo dT Primers
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    For cDNA synthesis using oligo dT primers in TurboCapture strips or plates Kit contents 100 l of oligo dT primers 0 4 g ul for cDNA synthesis with TurboCapture mRNA Kits
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    79237
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    Oligo dT Primers
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    Structured Review

    Qiagen oligo dt primer
    Oligo dT Primers
    For cDNA synthesis using oligo dT primers in TurboCapture strips or plates Kit contents 100 l of oligo dT primers 0 4 g ul for cDNA synthesis with TurboCapture mRNA Kits
    https://www.bioz.com/result/oligo dt primer/product/Qiagen
    Average 99 stars, based on 114 article reviews
    Price from $9.99 to $1999.99
    oligo dt primer - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia"

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq131

    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.
    Figure Legend Snippet: Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Techniques Used: Sequencing, Migration, Modification, Hybridization

    2) Product Images from "Determinants of RNA Quality from FFPE Samples"

    Article Title: Determinants of RNA Quality from FFPE Samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001261

    Fragmentation state and RT-PCR performance of RNA from tumor samples. RNA was isolated from individual 10 µm sections of FFPE tumor samples of different age (1–10 years). Fragmentation state was analysed by capillary electrophoresis (A). RNA performance was tested in 2-step real-time RT-PCR using assays directed against the human TBP gene, using either a mix of random and oligo-dT primers for cDNA synthesis (B), or oligo-dT alone (C).
    Figure Legend Snippet: Fragmentation state and RT-PCR performance of RNA from tumor samples. RNA was isolated from individual 10 µm sections of FFPE tumor samples of different age (1–10 years). Fragmentation state was analysed by capillary electrophoresis (A). RNA performance was tested in 2-step real-time RT-PCR using assays directed against the human TBP gene, using either a mix of random and oligo-dT primers for cDNA synthesis (B), or oligo-dT alone (C).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Formalin-fixed Paraffin-Embedded, Electrophoresis, Quantitative RT-PCR

    3) Product Images from "piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene"

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022727

    The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.
    Figure Legend Snippet: The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

    Techniques Used: Plasmid Preparation, Luciferase, Expressing, Transfection, Quantitation Assay, Quantitative RT-PCR, Western Blot

    4) Product Images from "Transcriptional Control of an Essential Ribozyme in Drosophila Reveals an Ancient Evolutionary Divide in Animals"

    Article Title: Transcriptional Control of an Essential Ribozyme in Drosophila Reveals an Ancient Evolutionary Divide in Animals

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004893

    D. melanogaster RPR co-purifies with RNase P and is required for its activity. A. RNase P activity was partially purified from D. melanogaster S2 cells using sequential DEAE- and SP-Sepharose (above). Pre-tRNA processing assays established that the peak of activity eluted in 300–500 mM NaCl (fractions 3–5). RNA isolated from all fractions was subjected to RT-PCR using RPR-specific primers. Amplicons corresponding to the expected RPR size were detected in fractions 3–5 that showed maximal RNase P activity. B. Thin-layer chromatographic analysis of RNase T2-cleaved tRNA Gly containing a 5′-pGp; the tRNA Gly was first generated from cleavage of internally [α- 32 P]-GTP-labeled pre-tRNA Gly by in vitro reconstituted E. coli RNase P or partially-purified D. melanogaster RNase P (lanes 1 and 4, respectively). The negative control (lane 3) shows RNase T2-cleaved internally labeled pre-tRNA Gly that lacks a 5′-pGp, and the positive control (lane 2) shows RNase T2-cleaved 5′-labeled pre-tRNA Gly that has a 5′-pGp. C. The predicted secondary structure of D. melanogaster RPR contains universally-conserved and functionally-important nucleotides (indicated by black circles). An antisense RNA oligonucleotide (red line; α-RPR-j7/2, complementary to a predicted single-stranded region between paired regions P7 and P2) was designed to inhibit RNase P activity. D. Partially-purified RNase P was inactivated with increasing concentrations of α-RPR-j7/2, but not with a scrambled oligo (sc-RPR-j7/2) that has the same nucleotide composition as α-RPR-j7/2. NC, negative control with no enzyme added; PC, positive control with in vitro reconstituted E. coli RNase P; IP, input; FT, flow-through.
    Figure Legend Snippet: D. melanogaster RPR co-purifies with RNase P and is required for its activity. A. RNase P activity was partially purified from D. melanogaster S2 cells using sequential DEAE- and SP-Sepharose (above). Pre-tRNA processing assays established that the peak of activity eluted in 300–500 mM NaCl (fractions 3–5). RNA isolated from all fractions was subjected to RT-PCR using RPR-specific primers. Amplicons corresponding to the expected RPR size were detected in fractions 3–5 that showed maximal RNase P activity. B. Thin-layer chromatographic analysis of RNase T2-cleaved tRNA Gly containing a 5′-pGp; the tRNA Gly was first generated from cleavage of internally [α- 32 P]-GTP-labeled pre-tRNA Gly by in vitro reconstituted E. coli RNase P or partially-purified D. melanogaster RNase P (lanes 1 and 4, respectively). The negative control (lane 3) shows RNase T2-cleaved internally labeled pre-tRNA Gly that lacks a 5′-pGp, and the positive control (lane 2) shows RNase T2-cleaved 5′-labeled pre-tRNA Gly that has a 5′-pGp. C. The predicted secondary structure of D. melanogaster RPR contains universally-conserved and functionally-important nucleotides (indicated by black circles). An antisense RNA oligonucleotide (red line; α-RPR-j7/2, complementary to a predicted single-stranded region between paired regions P7 and P2) was designed to inhibit RNase P activity. D. Partially-purified RNase P was inactivated with increasing concentrations of α-RPR-j7/2, but not with a scrambled oligo (sc-RPR-j7/2) that has the same nucleotide composition as α-RPR-j7/2. NC, negative control with no enzyme added; PC, positive control with in vitro reconstituted E. coli RNase P; IP, input; FT, flow-through.

    Techniques Used: Activity Assay, Purification, Isolation, Reverse Transcription Polymerase Chain Reaction, Generated, Labeling, In Vitro, Negative Control, Positive Control, Flow Cytometry

    Related Articles

    SYBR Green Assay:

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia
    Article Snippet: .. RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany). .. Data from surface antigen expression were set in relation to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which was found to be constantly expressed during vegetative growth and RDR silencing.

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    Article Snippet: .. Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Article Title: Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction
    Article Snippet: .. 1 μg of total RNA was reverse transcribed using an oligo-dT primer and afterwards analyzed by RT-QPCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the following primers: Actin forward: 5'-GGA CTT CGA GCA AGA GAT GG-3', Actin reverse: 5'-AGC ACT GTG TTG GCG TAC AG-3', Mcl-1 forward: 5'-TAA GGA CAA AAC GGG ACT GG-3', and Mcl-1 reverse: 5'-ACC AGC TCC TAC TCC AGC AA-3'. ..

    Article Title: Determinants of RNA Quality from FFPE Samples
    Article Snippet: .. Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) for cDNA synthesis either according to manufacturer's recommendations, or using oligo-dT primer in place of the supplied primer mix, followed by PCR using the QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Hilden, Germany). .. Thermal cycling conditions following the 15 minutes at 94°C activation step were: 15 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C for 40 cycles.

    Amplification:

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia
    Article Snippet: .. RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany). .. Data from surface antigen expression were set in relation to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which was found to be constantly expressed during vegetative growth and RDR silencing.

    Article Title: Transcriptional Control of an Essential Ribozyme in Drosophila Reveals an Ancient Evolutionary Divide in Animals
    Article Snippet: .. Reverse transcription and PCR cDNAs were prepared using an oligo dT primer (for mRNAs) or gene specific primers (for RPRs) by reverse transcription using an Omniscript RT kit (Qiagen). cDNAs were amplified with Taq DNA polymerase (NEB) using the recommended conditions and the following primer pairs: Forward DmelRPR, 5′-AGTCAGTTGCAAACTAGCATC-3′ and Reverse Dmel RPR, 5′- AGTCAGTCACAGATTAGTCTGAATTG-3′ ; Forward GFP, 5′-TAAGATATCATGGTGAGCAAGGG-3′ and Reverse GFP, 5′- ACCTCTAGATTACTTGTACAGCTCGTCC-3′ ; Forward Oda, 5′-GTCCTTCGGTAGAGCGACAT-3′ and Reverse Oda, 5′- GCACCATCTCGACTTCGTCT-3′ . .. Northern blot analysis D. melanogaster and D. virilis RPRs were detected using full-length anti-sense RNA probes labeled with [α-32 P]-ATP in an in vitro transcription reaction.

    Quantitative RT-PCR:

    Article Title: Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis
    Article Snippet: .. RT-PCR and qRT-PCR Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided. ..

    Article Title: Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction
    Article Snippet: .. 1 μg of total RNA was reverse transcribed using an oligo-dT primer and afterwards analyzed by RT-QPCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the following primers: Actin forward: 5'-GGA CTT CGA GCA AGA GAT GG-3', Actin reverse: 5'-AGC ACT GTG TTG GCG TAC AG-3', Mcl-1 forward: 5'-TAA GGA CAA AAC GGG ACT GG-3', and Mcl-1 reverse: 5'-ACC AGC TCC TAC TCC AGC AA-3'. ..

    Purification:

    Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis
    Article Snippet: .. Complementary DNA (cDNA) was reverse transcribed from total RNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using oligo-dT or random hexamer primers and purified using the Qiagen QiaQuick PCR purification kit. .. Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

    Real-time Polymerase Chain Reaction:

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    Article Snippet: .. Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Polymerase Chain Reaction:

    Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis
    Article Snippet: .. Complementary DNA (cDNA) was reverse transcribed from total RNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using oligo-dT or random hexamer primers and purified using the Qiagen QiaQuick PCR purification kit. .. Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia
    Article Snippet: .. RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany). .. Data from surface antigen expression were set in relation to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which was found to be constantly expressed during vegetative growth and RDR silencing.

    Article Title: Transcriptional Control of an Essential Ribozyme in Drosophila Reveals an Ancient Evolutionary Divide in Animals
    Article Snippet: .. Reverse transcription and PCR cDNAs were prepared using an oligo dT primer (for mRNAs) or gene specific primers (for RPRs) by reverse transcription using an Omniscript RT kit (Qiagen). cDNAs were amplified with Taq DNA polymerase (NEB) using the recommended conditions and the following primer pairs: Forward DmelRPR, 5′-AGTCAGTTGCAAACTAGCATC-3′ and Reverse Dmel RPR, 5′- AGTCAGTCACAGATTAGTCTGAATTG-3′ ; Forward GFP, 5′-TAAGATATCATGGTGAGCAAGGG-3′ and Reverse GFP, 5′- ACCTCTAGATTACTTGTACAGCTCGTCC-3′ ; Forward Oda, 5′-GTCCTTCGGTAGAGCGACAT-3′ and Reverse Oda, 5′- GCACCATCTCGACTTCGTCT-3′ . .. Northern blot analysis D. melanogaster and D. virilis RPRs were detected using full-length anti-sense RNA probes labeled with [α-32 P]-ATP in an in vitro transcription reaction.

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    Article Snippet: .. Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Article Title: Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction
    Article Snippet: .. 1 μg of total RNA was reverse transcribed using an oligo-dT primer and afterwards analyzed by RT-QPCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the following primers: Actin forward: 5'-GGA CTT CGA GCA AGA GAT GG-3', Actin reverse: 5'-AGC ACT GTG TTG GCG TAC AG-3', Mcl-1 forward: 5'-TAA GGA CAA AAC GGG ACT GG-3', and Mcl-1 reverse: 5'-ACC AGC TCC TAC TCC AGC AA-3'. ..

    Article Title: Determinants of RNA Quality from FFPE Samples
    Article Snippet: .. Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) for cDNA synthesis either according to manufacturer's recommendations, or using oligo-dT primer in place of the supplied primer mix, followed by PCR using the QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Hilden, Germany). .. Thermal cycling conditions following the 15 minutes at 94°C activation step were: 15 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C for 40 cycles.

    Random Hexamer Labeling:

    Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis
    Article Snippet: .. Complementary DNA (cDNA) was reverse transcribed from total RNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using oligo-dT or random hexamer primers and purified using the Qiagen QiaQuick PCR purification kit. .. Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

    Sequencing:

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    Article Snippet: .. Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Activated Clotting Time Assay:

    Article Title: Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction
    Article Snippet: .. 1 μg of total RNA was reverse transcribed using an oligo-dT primer and afterwards analyzed by RT-QPCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the following primers: Actin forward: 5'-GGA CTT CGA GCA AGA GAT GG-3', Actin reverse: 5'-AGC ACT GTG TTG GCG TAC AG-3', Mcl-1 forward: 5'-TAA GGA CAA AAC GGG ACT GG-3', and Mcl-1 reverse: 5'-ACC AGC TCC TAC TCC AGC AA-3'. ..

    Cellular Antioxidant Activity Assay:

    Article Title: Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction
    Article Snippet: .. 1 μg of total RNA was reverse transcribed using an oligo-dT primer and afterwards analyzed by RT-QPCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the following primers: Actin forward: 5'-GGA CTT CGA GCA AGA GAT GG-3', Actin reverse: 5'-AGC ACT GTG TTG GCG TAC AG-3', Mcl-1 forward: 5'-TAA GGA CAA AAC GGG ACT GG-3', and Mcl-1 reverse: 5'-ACC AGC TCC TAC TCC AGC AA-3'. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis
    Article Snippet: .. RT-PCR and qRT-PCR Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided. ..

    Article Title: Determinants of RNA Quality from FFPE Samples
    Article Snippet: .. Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) for cDNA synthesis either according to manufacturer's recommendations, or using oligo-dT primer in place of the supplied primer mix, followed by PCR using the QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Hilden, Germany). .. Thermal cycling conditions following the 15 minutes at 94°C activation step were: 15 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C for 40 cycles.

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    Qiagen oligo dt primers
    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with <t>Oligo</t> dT-primers, cDNA was analyzed by semi-quantitative <t>PCR</t> for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.
    Oligo Dt Primers, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt primers/product/Qiagen
    Average 99 stars, based on 290 article reviews
    Price from $9.99 to $1999.99
    oligo dt primers - by Bioz Stars, 2020-08
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    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Journal: BMC Genomics

    Article Title: Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

    doi: 10.1186/1471-2164-12-196

    Figure Lengend Snippet: KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Article Snippet: RT-PCR and qRT-PCR Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided.

    Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing, Marker, Polymerase Chain Reaction

    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    doi: 10.1093/nar/gkq131

    Figure Lengend Snippet: Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Article Snippet: RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany).

    Techniques: Sequencing, Migration, Modification, Hybridization

    Fragmentation state and RT-PCR performance of RNA from tumor samples. RNA was isolated from individual 10 µm sections of FFPE tumor samples of different age (1–10 years). Fragmentation state was analysed by capillary electrophoresis (A). RNA performance was tested in 2-step real-time RT-PCR using assays directed against the human TBP gene, using either a mix of random and oligo-dT primers for cDNA synthesis (B), or oligo-dT alone (C).

    Journal: PLoS ONE

    Article Title: Determinants of RNA Quality from FFPE Samples

    doi: 10.1371/journal.pone.0001261

    Figure Lengend Snippet: Fragmentation state and RT-PCR performance of RNA from tumor samples. RNA was isolated from individual 10 µm sections of FFPE tumor samples of different age (1–10 years). Fragmentation state was analysed by capillary electrophoresis (A). RNA performance was tested in 2-step real-time RT-PCR using assays directed against the human TBP gene, using either a mix of random and oligo-dT primers for cDNA synthesis (B), or oligo-dT alone (C).

    Article Snippet: Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) for cDNA synthesis either according to manufacturer's recommendations, or using oligo-dT primer in place of the supplied primer mix, followed by PCR using the QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Hilden, Germany).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Formalin-fixed Paraffin-Embedded, Electrophoresis, Quantitative RT-PCR

    The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

    Journal: PLoS ONE

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene

    doi: 10.1371/journal.pone.0022727

    Figure Lengend Snippet: The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

    Article Snippet: Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions.

    Techniques: Plasmid Preparation, Luciferase, Expressing, Transfection, Quantitation Assay, Quantitative RT-PCR, Western Blot